Hepatitis C Virus (HCV) is recognized as a worldwide health problem affecting over 170 million people. HCV causes a spectrum of disease ranging from an asymptomatic carrier state to end-stage liver disease; which includes cirrhosis and hepatocellular carcinoma. A vaccine for HCV is not available. The present therapies for HCV infection do not often result in viral clearance, are difficult to administer and result in serious toxic side effects. The development of new therapeutic agents for HCV infection is a major public health priority. Studies on HCV are challenging due to its inefficient replication in cell culture. The HCV replicon system, based on the self-replication of engineered mini- genomes in cell culture, has proven invaluable for studying HCV genomic replication, translation and polyprotein processing. Utilizing the replicon system, the HCV-encoded serine protease (NS3) and RNA polymerase (NS5B) have emerged as favorite pharmaceutical targets. However, this system does not support the production of HCV particles or allow for the study of viral entry, the first event in the HCV lifecycle. Our strategy is to develop anti-HCV agents that will block novel non-enzymatic stages of the viral replicative cycle, specifically the viral host cell entry mechanisms. The selective association of a virus with a target cell is determined by an interaction between viral glycoproteins and specific cell-surface receptor(s) and is essential in the initiation of infection. HCV encodes two envelope glycoproteins, E1 and E2, which accumulate in the endoplasmic reticulum, the proposed site for HCV assembly and budding. In the absence of a suitable native HCV model, pseudotype viruses have been developed as surrogate models to study virus attachment/entry. These model systems allow the study of viral entry into the cell but do not replicate other aspects of the viral life cycle. Significant advancements have been made over the years in (i) the characterization of envelope glycoproteins and (ii) understanding the functional role for the ectodomains of E1 and E2 glycoproteins in the recognition of host cell surface molecules. Furthermore, HCV has a high mutation rate and the emergence of drug- resistant virus is highly likely. Therefore, we believe that inhibition of HCV entry is a promising approach, which will complement other mechanistic approaches. Our objective is to discover and develop HCV entry inhibitors for the treatment of HCV infection. In Phase I we will develop a prototype high-throughput assay utilizing a HIV derived HCV pseudotype virus, containing a luciferase reporter gene, to measure virus infection. This assay will become a valuable tool for screening libraries of structurally diverse small molecules, in order to identify potent inhibitors of HCV entry. We will confirm antiviral activity in secondary HCV pseudotype infection plaque assays. Lead compounds will be evaluated for activity across all genotypes. In Phase II, we will progress the most active scaffolds through a rational drug design program and lead compounds will be tested for efficacy and toxicity in animal models. The most promising compound will advance to IND-enabling toxicology and pharmacology studies in two species (Phase III). [unreadable] [unreadable]